Red to near-infrared photobiomodulation treatment of the visual system in visual system disease or injury

ABSTRACT

A method of treating visual system disease is disclosed. One embodiment comprises the steps of (a) exposing a component of a patient&#39;s visual system to light treatment, wherein the light treatment is characterized by wavelength of between 630-1000 nm and power intensity between 10-90 mW/cm 2  for a time of 1-3 minutes, and (b) observing restoration of visual system function.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. provisional application60/440,816, filed Jan. 17, 2003, incorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with United States government support awarded bythe following agencies: Defense Advanced Research Projects Agency GrantDARPA N66001-01-1-8969 and N66001-03-1-8906, National Institute ofEnvironmental Health Sciences Grant ES06648, National Eye Institute CoreGrant P30-EY01931, National Eye Institute Grants EY11396 and EY05439.The United States has certain rights in this invention.

BACKGROUND OF THE INVENTION

Decrements in mitochondrial function have been postulated to be involvedin the pathogenesis of numerous retinal and optic nerve diseases,including age-related macular degeneration, diabetic retinopathy, andLeber's hereditary optic neuropathy (J. F. Rizzo, Neurology 45:11-16,1995; M. J. Baron, et al., Invest. Ophthalmol. Visual Sci. 42:3016-3022,2001; V. Carelli, et al., Neurochem. Int. 40:573-584, 2002). Decrementsin mitochondrial function have also been postulated to be involved inthe pathogenesis in methanol intoxication (M. M. Hayreh, et al.,Neurotoxicity of the Visual System, eds. Merigan, W. & Weiss, B. (Raven,New York), pp. 35-53, 1980; G. Martin-Amat, et al., Arch. Ophthalmol.95:1847-1850, 1977; M. T. Seme, et al., J. Pharmacol. Exp. Ther.289:361-370, 1999; M. T. Seme, et al., Invest. Ophthalmol. Visual Sci.42:834-841, 2001). Methanol intoxication produces toxic injury to theretina and optic nerve, frequently resulting in blindness. A toxicexposure to methanol typically results in the development of formicacademia, metabolic acidosis, visual toxicity, coma, and, in extremecases, death (J. T. Eells, Browning's Toxicity and Metabolism ofIndustrial Solvents: Alcohols and Esters, eds. Thurman, T. G. Kaufmann,F. C. (Elsevier, Amsterdam), Vol. IV, pp. 3-15, 1992; R. Kavet and K.Nauss, Crit. Rev. Toxicol. 21:21-50, 1990). Visual disturbancesgenerally develop between 18 and 48 hours after methanol ingestion andrange from misty or blurred vision to complete blindness. Both acute andchronic methanol exposure have been shown to produce retinal dysfunctionand optic nerve damage clinically (J. T. Eells, supra, 1992; R. Kavetand K. Nauss, supra, 1990; J. Sharpe, et al., Neurology 32:1093-1100,1982) and in experimental animal models (S. O. Ingemansson, ActaOphthalmol. 158(Supp):5-12, 1983; J. T. Eells, et al., Neurotoxicology21:321-330, 2000; T. G. Murray, et al., Arch. Ophthalmol. 109:1012-1016,1991; E. W. Lee, et al., Toxicol. Appl. Pharmacol. 128:199-206, 1994).

Formic acid is the toxic metabolite responsible for the retinal andoptic nerve toxicity produced in methanol intoxication (M. M. Hayreh, etal., supra, 1980; G. Martin-Amat, et al., supra 1977; M. T. Seme, etal., supra, 1999; M. T. Seme, supra, 2001; G. Martin-Amat, et al.,Toxicol. Appl. Pharmacol. 45:201-208, 1978). Formic acid is amitochondrial toxin that inhibits cytochrome c oxidase, the terminalenzyme of the mitochondrial electron transport chain of all eukaryotes(P. Nicholls, Biochem. Biophys. Res. Commun. 67:610-616, 1975; P.Nicholls, Biochim. Biophys. Acta430:13-29, 1976). Cytochrome oxidase isan important energy-generating enzyme critical for the properfunctioning of almost all cells, especially those of highly oxidativeorgans, including the retina and brain (M. T. T. Wong-Riley, TrendsNeurosci. 12:94-101, 1989). Previous studies in our laboratory haveestablished a rodent model of methanol-induced visual toxicity anddocumented formate-induced mitochondrial dysfunction and retinalphotoreceptor toxicity in this animal model (M. T. Seme, et al., supra,1999; M. T. Seme, et al., supra, 2001; J. T. Eells, et al., supra, 2000;T. G. Murray, et al., supra, 1991).

Photobiomodulation by light in the red to near-IR range (630-1,000 nm)using low-energy lasers or light-emitting diode (LED) arrays has beenshown to accelerate wound healing, improve recovery from ischemic injuryin the heart, and attenuate degeneration in the injured optic nerve (H.T. Whelan, et al., J. Clin. Laser Med. Surg. 19:305-314, 2001; U. Oron,et al., Lasers Surg. Med. 28:204-211, 2001; E. M. Assa, et al., BrainRes. 476:205-212, 1989; M. J. Conlan, et al., J. Clin. Periodont.23:492-496, 1996; W. Yu, et al., Lasers Surg. Med. 20:56-63, 1997; A. P.Sommer, et al., J. Clin. Laser Med. Surg. 19:27-33, 2001). At thecellular level, photoirradiation at low fluences can generatesignificant biological effects, including cellular proliferation,collagen synthesis, and the release of growth factors from cells (M. J.Conlan, et al., supra, 1996; T. Karu, J. Photochem. Photobiol. 49:1-17,1999; M. C. P. Leung, et al., Lasers Surq. Med. 31:283-288, 2002). Ourprevious studies have demonstrated that LED photoirradiation at 670 nm(4 J/cm²) stimulates cellular proliferation in cultured cells andsignificantly improves wound healing in genetically diabetic mice (H. T.Whelan, et al., supra, 2001; A. P. Sommer, et al., supra, 2001). Despiteits widespread clinical application, the mechanisms responsible for thebeneficial actions of photobiomodulation have not been elucidated.Mitochondrial cytochromes have been postulated as photoacceptors for redto near-IR light energy and reactive oxygen species have been advancedas potential mediators of the biological effects of this light (Karu,supra, 1999; N. Grossman, et al., Lasers Surg Med. 22:212-218, 1998).

BRIEF SUMMARY OF THE INVENTION

In one embodiment, the present invention is a method of treating diseaseor injury of the visual system, comprising the steps of (a) exposing acomponent of a patient's visual system to light treatment, wherein thelight treatment is characterized by wavelength between 630-1000 nm andpower intensity between 10-90 mW/cm² for a time of 1-3 minutes, and (b)observing restoration or protection of visual system function.Preferably, the wavelength is selected from the group consisting of 670nm, 830 nm and 880 nm.

In one embodiment of the invention, the light treatment is characterizedby an energy density of between 0.5-20 J/cm². In a preferred embodiment,the energy density is between 2-10 J/cm².

Preferably, the patient is exposed to light treatment multiple times andis exposed to light treatment intervals of 24 hours.

In a preferred form of the invention, the component of the visual systemcomprises the patient's retina or is selected from the group consistingof cornea and optic nerve.

Other features, objects or advantages of the present invention willbecome apparent to one of skill in the art after examination of thespecification, claims and drawings.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a graph of wavelength versus relative absorbance, content oractivity.

FIG. 2 is a graph of rod and M-cone ERG amplitude (μV) versus logrelative retinal illumination.

FIG. 3 is a graph of UV-cone ERG amplitude (μV) versus log relativeretinal illumination.

FIG. 4(A-D) is a set of micrographs illustrating outer retinalmorphology in representative untreated control (FIG. 4A), LED control(FIG. 4B), methanol-intoxicated (FIG. 4C), and LED-treated,methanol-intoxicated (FIG. 4D) retinas.

FIG. 5(A-D) is a set of electron micrographs of the rod inner segmentregion in representative untreated control (FIG. 5A) LED control, (FIG.5B) methanol-intoxicated, (FIG. 5C) and LED-treated,methanol-intoxicated (FIG. 5D) rats.

FIG. 6(A-B) is a set of graphs. FIG. 6A is a graph of rod and M-cone ERGamplitude versus log relative retinal illumination. FIG. 6B is a graphof ERG amplitude versus log relative retinal illumination.

FIGS. 7A and B describes NIR LED treatment as improving healingfollowing laser-induced retinal injury. FIG. 7A is a set of micrographsof a laser grid at 15 minutes and 1 month post-laser treatment in bothLED-treated and non-LED-treated tissue. FIG. 7B is a bar graph ofseverity of burn (spot persistence) versus treatment.

FIG. 8 describes NIR LED treatment as improving visual function. FIG. 8Ais a set of micrographs of lateral geniculate nuclei of monkeys withmonocular central retinal laser injury both treated and not treated withNIR-LED. FIG. 8B is a bar graph of untreated and LED-treated subjectsversus percentage of metabolically active neurons in layer 6 of LGN.

FIG. 9 describes NIR-LED treatment as improving retinal function. FIG. 9is a graph of the multifocal ERG response in nanovolts versus pre-laser,post-laser, four day post-laser and eleven day post-laser treatment forLED-treated and non-treated tissue.

DETAILED DESCRIPTION OF THE INVENTION

Low energy photon irradiation by light in the far red to near infraredspectral (range 630-1000 nm) using low energy lasers or light emittingdiode arrays has been found to modulate various biological processes incell culture and animal models [(M. J. Conlan, et al., J. Clin.Periodont. 23:492-496, 1996; W. Yu, et al., Lasers Surg. Med. 20:56-63,1997; A. P. Sommer, et al., J. Clin. Laser Med. Surg. 19:27-33, 2001; T.Karu, J. Photochem. Photobiol. 49:1-17, 1999)]. As described above, thisphenomenon of photobiomodulation has been applied clinically in thetreatment soft tissue injuries and to accelerate wound healing [(H. T.Whelan, et al., J. Clin. Laser Med. Surg. 19:305-314, 2001; U. Oron, etal., Lasers Surg. Med. 28:204-211, 2001; E. M. Assa, et al., Brain Res.476:205-212, 1989; M. J. Conlan, et al., supra, 1996)]. The mechanism ofphotobiomodulation by red to near infrared light at the cellular levelhas been ascribed to the activation of mitochondrial respiratory chaincomponents resulting in initiation of a signaling cascade which promotescellular proliferation and cytoprotection.

The therapeutic effects of red to near infrared light may result, inpart, from the stimulation of cellular events associated with increasesin cytochrome c oxidase activity. In support of this theory, we havedemonstrated in primary neuronal cells that LED photobiomodulation (670nm at 4 J/cm²) reverses the reduction in cytochrome oxidase activityproduced by the blockade of voltage-dependent sodium channel function bytetrodotoxin (M. T. T. Wong-Riley, et al., NeurReport 12:3033-3037,2001). In addition, we have shown that the action spectrum for LEDstimuation of cytochrome oxidase activity and cellular ATP contentparallels the absorption spectrum for cytochrome oxidase. (FIG. 1). Thestructured nature of the action spectrum is strong evidence thatcytochrome oxidase is a primary photoacceptor molecule for light in thefar red to near infrared region of the spectrum. Our recent work hasprovided evidence for the therapeutic benefit of photobiomodulation inthe survival and functional recovery of the retina and optic nerve invivo after acute injury by the mitochondrial toxin, formic acidgenerated in the course of methanol intoxication. (Eells, et al., Proc.Natl. Acad. Sci. 100(6):3439-3441, incorporated by reference herein.) Inaddition, we have provided data below indicating that 670 nm LEDtreatment promotes retinal healing and improved visual functionfollowing high intensity laser-induced retinal injury in adult non-humanprimates.

These findings provide a link between the actions of red to nearinfrared light on mitochondrial oxidative metabolism in vitro and ocularinjury in vivo. Importantly, the results of these studies and otherssuggested to us that photobiomodulation with red to near infrared lightaugments recovery pathways promoting neuronal viability and restoringneuronal function following ocular injury. There was no evidence ofdamage to the retina or optic nerve following 670 nm LED treatment.Based on these findings, we suggest that photobiomodulation represents anon-invasive and innovative therapeutic approach for the treatment ofocular injury and acute and chronic ocular disease.

In one broad aspect, the present invention is a method of treatingocular disease comprising the steps of exposing a component of apatient's visual system to light treatment wherein the light treatmentis characterized by wavelength between 630-1000 nm and power intensitybetween 25-50 mW/cm² for a time of 1-3 minutes (equivalent to an energydensity of 2-10 J/cm²) and observing restoration or protection of visualfunction. The sections below further describe and characterize thepresent invention.

Suitable LED

In a preferred form of the present invention, the therapeuticphotobiomodulation is a achieved using an light emitting diode as thesource of red to near infrared light unlike the prior art which utilizedlight generated by low energy laser. Lasers have limitations in beamwidth, wavelength capabilities, and size of the injury or tissue thatcan be treated. In addition, heat generated from laser light can damagebiological tissue, and the concentrated beam of laser light mayaccidentally damage the eye. Light-emitting diode (LED) arrays weredeveloped for the National Aeronautics and Space Administration mannedspace flight experiments. In comparison with lasers, LED technologygenerates negligible amounts of heat, is clinically proven to be safe,and has achieved non-significant risk status for human trials by theU.S. Food and Drug Administration.

Preferably, the present inventions utilizes a noncoherent light sourcecapable of irradiating the entire retina and optic system withmonochromatic light in the far-red to near-infrared region of thespectrum. An example of a commercially available, preferable LED sourceis the ISO 9001 LED (QUANTUM SPECTRALIFE) array obtainable from QuantumDevices (Barneveld, Wis.).

Light in the far-red to near infrared region of the spectrum is known topenetrate nearly 20 cm into irradiated tissue. A preferable device iscomposed of a monolithic array of hybrid GaAIAs light emitting diodesdesigned to emit diffused monochromatic light. Preferable LED chips havebeen fabricated to emit specific peak wavelengths (between 650-940 nm)of photon energy and the system has been designed to deliver highintensity light energy to an isolated area of exposure without heat.

Preferably, the LEDs are shielded by a glass barrier and are unlensed,allowing for even dispersion of light over a 180° viewing angle allowingeach LED chip to act as a point source offering a high degree ofillumination uniformity. The surface energy (photon flux or powerintensity) delivered by the LED units is between 25-50 mW/cm². LED unitsthat we have worked with produce monochromatic light at wavelengths inthe far red (670 nm) to near infrared (700-900 nm) region of thespectrum. The LED arrays can be custom fabricated from GaAIAs (galiniumaluminum arsinate) diodes to produce light with peak wavelengths between650 nm and 940 nm. The devices that we have employed have peakwavelengths of 670, 728, 830 and 880 nm with bandwidths of 25-35 nm at50% power. LED units and low energy lasers could be constructed thatemit at other peak wavelengths in the range of 630-1000 nm.

Peak wavelengths of 670 nm, 830 nm, and 880 nm with bandwidths of 25-30nm have been used with success in experimental and clinical studies.Both benchtop LED units (bandwidth 25-30 cm at 50% power) with 8×10 cmrectangular arrays and portable NIR-LED units with 5 cm diametercircular arrays obtained from Quantum Devices, Inc. (Barneveld, Wis.)have been used in our experimental and clinical studies. In ourexperimental and clinical studies of NIR-LED treatment of ocular injuryor disease, 670 nm LED irradiation was administered at a power intensitybetween 25-50 mW/cm² between 1-3 minutes to produce an energy density of4 J/cm².

In a preferred embodiment of the present invention, 670 nm LEDirradiation administered at a power intensity between 25-50 mW/cm²between 1-3 minutes produces an energy density of 4 J/cm² and promotesretinal healing and improves visual function. It is likely that otherNIR wavelengths (830, 880 nm) will also promote retinal healing andprotect against retinal injury, but we have no animal model evidence forthis at this time.

The LED unit is typically positioned at a distance of approximately 2.5cm from the eye in each case. We envision that one would wish toposition the light source at between 0.5 cm and 4.0 cm from the eye or0.5-1.0 cm from the top, side or back of the head for irradiation ofother components (optic nerve, lateral geniculate nucleus, superiorcolliuculus, etc.) of the visual system. As described below, theposition from the target factors into the entire energy densitycalculation.

Optimal Treatment Protocols

There are four important treatment parameters variables in the therapyof the present invention: (1) Energy density or fluence, (2) lightwavelength (3) number of treatments and (4) the treatment interval.

(1) Energy density or fluence is the product of LED power intensity andduration of irradiation and is expressed as Joules per square centimeter(J/cm²). For effective NIR-light therapy of the present invention, theenergy density cannot be too low, otherwise there will be no biologicaleffect. Energy density should also not be too great or it might produceadverse effects. Prior studies have suggested that biostimulation occursat energy densities between 0.5 and 20 J/cm², whereas energy densitiesabove 20 J/cm² exert bioinihibitory effects. Preferable energy densityof the present invention is between 0.5-20 J/cm², most preferablybetween 2-10 J/cm². This range is based on evidence which documents thatexposure to near-infrared light at energy densities (fluence) between2-10 J/cm² promotes cellular energy metabolism, cell division and woundhealing, protects against toxin-induced retinal damage and promoteshealing and improved visual function following high intensitylaser-induced retinal injury.

(2) With respect to LED wavelength, the majority of our studies havebeen conducted using 670 nm LED light and there is substantial evidencethat NIR-LED treatment at 670 nm is beneficial for the treatment ofocular toxicity, retinal laser injury and retinal disease. NIR-LED lightat wavelengths corresponding to the absorption peaks of the coppercenters in the cytochrome oxidase molecule (670 nm, 830 nm and 880 nm)have been shown to be effective in promoting the recovery of cytochromeoxidase activity and energy metabolism in cultured primary neurons.These three wavelengths are preferable wavelengths in the presentinvention. (M. Wong-Riley, et al., EPEC Conference, EuropeanBioenergetics Conference, 2002)

Band width can vary depending on technology and type of light sourceused. Although LED arrays are preferred in the present invention, theinvention includes any far-red to near infrared light source (low energylasers and LED arrays) which can produce energy densities between 0.5-20J/cm². Preferable bandwidth for low energy laser light sources would be4 nm and preferable band width for LEDs would be 25-50 nm. The entirepreferred range would be 4-50 nm.

(3) In acute ocular injury situations, the optimal time for initialNIR-LED treatment should be within 24 hours of injury, if possible,based on our studies of acute ocular injury in rodent and primatemodels. However, if treatment is not feasible within the first 24 hours,it should be initiated as soon as possible. Molecular studies documentupregulation of genes encoding energy producing and antioxidant proteinswithin 24 hours of NIR-LED treatment.

In summary, a preferred form of the present invention uses near infraredwavelengths of 630-1000, most preferably, 670-900 nm (bandwidth of 25-35nm) with an energy density exposure of 0.5-20 J/cm², most preferably2-10 J/cm², to produce photobiomodulation. This is accomplished byapplying a target dose of 10-90, preferably 25-50 mW/cm² LED generatedlight for the time required to produce that energy density. Timerequirements are calculated as: Power-intensity (mW/cm²)×Time(seconds)÷1,000=Energy Density (J/cm²).

(4) Treatment intervals of 24 hours have been shown to be beneficial forocular injuries. Other studies have documented efficacy with treatmentsadministered 2-3 times per day. It is likely that treatment spaced 2-3days apart may also be effective. For chronic diseases, NIR-LEDtreatments administered at weekly intervals may be beneficial.

We suggest a treatment regime as described below, at 5, 25 and 50 hours.We suggest that the treatment be within 1-3 minutes of duration toprovide the appropriate energy density.

(5) Number of treatments: We have demonstrated success in treatment ofacute ocular injuries with as few as 2, preferably 3 or more, treatmentsand as many as 21 treatments administered at 1 day intervals. For thetreatment of chronic diseases, NIR-LED treatments may be administeredindefinitely.

Suitable Treatment Methods

In a preferred embodiment of the present invention, one would expose anycomponent of a patient's visual system to the therapeutic effects of thelight treatment described above. By “visual system” we mean to includethe cornea, iris, lens, retina, optic nerve, optic chiasm, lateralgeniculate nucleus, superior colliculus, pretectal nucleic, theaccessory optic system, the oculomotor system, pulvinar, opticradiations, visual cortex, and associational visual cortical areas.

Exposure of the visual system may occur by treating with light directedinto the eyes, thus irradiating the cornea, lens, iris, retina and opticnerve head. Alternatively, the device can be oriented so that the lightis directed through the back of the head, thus irradiating the visualcortex or through the sides or top of the head thus irradiating theother components of the visual system.

One would wish to observe restoration or protection of visual functionas measured in any conventional way that assesses visual function.

Therapeutic endpoints for treatment of corneal abrasion would includeabsence of fluorescein staining of the cornea. For retinal injury ordisease, therapeutic endpoint measurement would include: (1) Fundoscopyor fundus photography which is an assessment of the appearance of thefundus or back of the eye, (note that the retina and optic nerve areobserved by using special lenses); (2) Optical coherence tomographywhich measures the thickness (cross sectional architecture) of theretina; (3) Flash, flicker or multifocal electroretinogram recordingswhich measure the electrical response of the rod and cone photoreceptorsin the retina to a light stimulus; (4) The visual evoked corticalpotentials which access the integrity of the retino-geniculo striatepathway by measuring the electrical response of the visual area of thebrain recorded from scalp electrodes to color vision testing; and (5)Visual acuity assessment using optotype (Snellen-style) eye charts. Onewould expect to see improvement or protection of the retina as measuredby the methods described above.

For the optic nerve, therapeutic endpoint measurement would include themeasurement of the visual evoked cortical potential from regions of theLGN or superior colliculus, to which the optic nerves project and thePupillary Light Reflex test, which tests the integrity of the opticnerve (cranial nerve 2) and the oculomotor nerve (cranial nerve 3).

Therapeutic endpoints for improvement of visual function (measuring LEDimprovement of disease or injury to other components of the visualsystem—optic nerve, LGN visual pathways, etc.) preferably involves theuse of a battery of tests which serve as standardized assessments forevaluation of the visual functions important in ensuring that visualperceptual processing is accurately completed. These include assessmentof visual acuity (distant and reading), contrast sensitivity function,visual field, oculomotor function visual attention and scanning.

More detailed descriptions of retinal and visual function tests include:

1. Kinetic (Goldmann) perimetry (“Perimetry” is the quantitative testingof the side vision).

2. Automated (computerized) perimetry. In this test, spots of light areautomatically projected into predetermined areas of the visual field.The test continues until the dimmest light is found that can be seen ineach area of the side vision. These visual field tests provide importantinformation.

3. Critical Flicker Fusion Frequency (CFF). Patients view a flickeringlight to test the ability of the optic nerve to conduct impulses withuniform speed. This test has proven to be very useful in identifyingvisual loss due to optic nerve damage.

4. Infra-red video pupillography. This is a way of seeing the pupilsclearly in the dark so that a more certain diagnosis can be made. Wealso use it to transilluminate the iris to identify local iris causesfor pupillary abnormalities.

5. Electroretinography. A regular ERG (eletroretinogram) records theelectrical activity of the whole retina in response to light and helpsto tell us if the rods and cones of the retina are firing in the correctway.

6. The Multi-focal ERG (MERG) does about a hundred ERGs at once byilluminating various little bits of the retina sequentially. It uses acomputer to sort out the dizzying torrent of information and then itpresents a map of the sensitivity of various parts of the retina, basedon the electrical activity (in response to light) of all those differentregions. If this map matches the map from perimetry, then the problem isin the retina and not in the optic nerve or brain.

7. Multi-focal Visual-Evoked Potentials (MVEP). Using a MERG stimulus,information can be picked up from the scalp that tells us if the visualpathways in the brain are damaged.

8. Computer controlled infra-red sensitive pupillography. This method isused to monitor pupillary movements in response to different types oflight in order to quantify how much damage there might be in the visualsystem.

9. Computer controlled “Pupil” Perimetrv. This method uses the pupilmovement in response to small lights presented in the field of vision asan objective indicator of how well the eye sees the light.

10. Computer recording of eye movements. This instrument can be used formonitoring pupil movements—but it also has the capacity to record thesmall movements of both eyes at the same time to see if they aretracking together and have normal movements in different directions ofgaze.

11. Optical Coherence Tomography (OCT). This is a new device that looksat the retina at the back of the eye and measures the thickness of thelayer of nerves coming from all quadrants of the retina and leading intothe optic nerve. This nerve fiber layer may be thickened, thinned ornormal, depending on the nature of the disease affecting the opticnerve.

12. Ishihara Color Vision Test Cards. Used for color vision evaluation.A test chart on color dots that appear as identifiable numbers orpatterns to individuals who have various types of color vision deficits.

The retina is a complex sensory organ composed of different cell typesarranged in distinct layers. The term “retinal function” will be used torefer to (1) activation of these layers by a light stimulus and (2) theprocesses required for maintenance of the cell. Different diseases mayaffect the retinal layers or cell types in a selective fashion.Congenital stationary night blindness affects transmission of visualsignals in the rod-mediated visual pathway whereas achromatopsia affectsonly the cone pathway. Other diseases may affect both photoreceptortypes in a defined location on the retina. Examples are the maculardystrophies, such as Stargardt's and age-related macular degeneration.Other diseases, such as glaucoma or optic neuropathy appear to affectprimarily the ganglion cells, located on the surface of the innerretina.

Assessment of the efficacy of a therapeutic intervention in one of theseretinal diseases therefore depends on the specific disorder. Congenitalstationary nightblindness would be best assessed by the full-fieldelectroretinogram in a patient that has been adapted to darkness forabout 30 minutes. Conversely achromatopsia, absent cone function, isbest assessed by a full-field electroretinogram under light-adaptedconditions and with a rapidly flickering flash stimulus that isolatescone function. Diseases of the macula are evident in the multifocal ERG,but not the full-field. This is due to the fact the macula, with severalhundred thousand photoreceptors makes a very small contribution to thefull-field ERG signal, which is the sum of 12 million or morephotoreceptors. For this reason, assessment of the therapeutic efficacyof an intervention to treat Stargardt's disease or age-related maculardegeneration, would be best accomplished by the multifocal ERG. Neitherfull-field ERGs nor multifocal ERGs contain a significant contributionfrom the ganglion cell layer. Assessment of interventions to affect theprogression of glaucoma or Leber's hereditary optic neuropathy thus usethe visually-evoked cortical potential because the visual corticalresponse is wholly-dependent on ganglion cell function and because theERG is not affected in these diseases.

The summary, there are a number of different tests used in clinicalophthalmology that are designed to objectively measure the function ofthe retina. The retina must perform a number of jobs in order to converta quantum of light entering the eye into an action potential in thevisual cortex. The activation of the retinal layers by light results inthe generation of electric fields in various levels of the visual systemthat can be recorded non-invasively. In theory, the NIR therapy could bebeneficial in a wide range of diseases since it appears to affect basiccellular responses to insult such as ATP production and apoptosis. Thusthere would be no one test that would be appropriate to assessing allthe diseases that might benefit for NIR therapy.

For further assessment information, one may wish to consult AmericanOptometric Association (AOA), Comprehensive adult eye and visionexamination: Reference Guide for Clinicians. St. Louis (Mo.): AmericanOptometric Association (AOA); 1994; Clinical Ophthalmology: A SystematicApproach by Jack J. Kanski, et al., Butterworth-Heinemann Medical; 5thedition (Jun. 2, 2003fe); A Textbook of Clinical Ophthalmology, 2ndEdition; and A Practical Guide to Disorders of the Eyes and TheirManagement by Ronald Pitts Crick (King's College Hospital, London) andPeng Tee Khaw (Moorfields Eye Hospital, London); and NoninvasiveDiagnostic Techniques in Ophthalmology Barry R. Masters (Editor), ISBN:0387969926, Pub. Date: August 1990 Publisher: Springer-Verlag New York,Incorporated.

Treatment Candidates

Numerous ocular diseases and injuries are likely to benefit from NIR-LEDtherapy. These include: 1) Acute ocular injuries to the cornea, retinaand optic nerve, such as corneal abrasions, acute retinal ischemia,retinal detachment, light or laser induced retinal injuries. 2)Intoxications affecting the visual system following exposure to, oringestion of, environmental toxins (e.g. methanol, pesticides) and drugs(e.g. ethambutal). 3) Chronic retinal and optic nerve diseasesincluding, but not limited to, glaucoma, age-related maculardegeneration, diabetic retinopathy, Leber's hereditary optic neuropathy,and other mitochondrial diseases with ocular manifestations. 4)Retinopathies and optic neuropathies resulting from nutritionaldeficiencies (e.g. folate, vitamin B₁₂). 5) Lesions of any centers inthe visual system, including the optic nerve, optic chiasm, opticradiation, dorsal lateral geniculate nucleus, superior colliculus, andthe visual cortex. Lesions could be induced by accident, trauma,hemorrhage, blood clot, ischemia, tumor, inflammation, infection orgenetic defects.

EXAMPLES Example 1 LED Treatment Protects the Rat Retina fromHistopathic Changes Induced by Methanol-Derived Formate.

In General

We hypothesize that the therapeutic effects of red to near-IR lightresult, in part, from the stimulation of cellular events associated withincreases in cytochrome c oxidase activity. In support of thishypothesis, we have recently demonstrated in primary neuronal cells thatLED photobiomodulation (670 nm at 4 J/cm²) reverses the reduction incytochrome oxidase activity produced by the blockade ofvoltage-dependent sodium channel function by tetrodotoxin (M. T. T.Wong-Riley, et al., NeuroReport 12:3033-3037, 2001). The present studiesextended these investigations to an in vivo system to determine whether670-nm LED treatment would improve retinal function in an animal modelof methanol-induced mitochondrial dysfunction.

Using the electroretinogram (ERG) as a sensitive indicator of retinalfunction, we demonstrated that three brief (2 minutes, 24 seconds)670-nm LED treatments 4 J/cm²) delivered 5, 25, and 50 hours after theinitial dose of methanol attenuated the retinotoxic effects ofmethanol-derived formate. Our studies demonstrate a significant recoveryof rod- and M-cone mediated retinal function as well as a significantrecovery of UV-cone mediated function in LED-treated rats. We furthershow that LED treatment protected the retina from methanol-inducedhistopathology. The present study provides evidence that 670 nm LEDtreatment promotes the recovery of retinal function and protects theretina against the cytotoxic actions of the mitochondrial toxin, formicacid. Our findings are consistent with hypothesis that LEDphotobiomodulation at 670 nm improves mitochondrial respiratory chainfunction and promotes cellular survival in vivo. They also suggest thatphotobiomodulation may enhance recovery from retinal injury and fromother ocular diseases in which mitochondrial dysfunction is postulatedto play a role.

Methods

Materials. LED arrays (8×10 cm) were obtained from Quantum Devices(Barneveld, Wis.). Methanol (HPLC grade) obtained from Sigma was dilutedin sterile saline and administered as a 25% (wt/vol) solution.Thiobutabarbitol sodium salt (Inactin) was purchased from ResearchBiochemicals (Natick, Mass.). Atrpine sulfate was obtained from AmVetPharmaceuticals (Fort Collins, Colo.). Hydroxypropyl methylcellulose(2.5%) drops were acquired from IOLAB Pharmaceuticals, Claremont, Calif.Atropine sulfate ophthalmic solution drops were purchased from PhoenixPharmaceutical (St. Joseph, Mo.). All other chemicals were reagent gradeor better.

Animals. Male Long-Evans rats (Harlan Sprague-Dawley, Madison, Wis.),which weighed 250-350 g, were used throughout these experiments. Allanimals were supplied food and water ad libitum and maintained on a 12hour light/dark schedule in a temperature- and humidity-controlledenvironment. Animals were handled in accordance with the Guide for theCare and Use of Laboratory Animals as adopted and promulgated by theNational Institutes of Health.

Methanol-Intoxication Protocol. Animals were randomly assigned to one offour treatment groups: (1) Untreated control, (2) LED-treated control,(3) methanol-intoxicated and (4) LED-treated methanol- intoxicated rats.Rats were placed in a thermostatically controlled plexiglass chamber(22×55×22 cm; maintained at 22-23° C.) and exposed to a mixture ofN₂O/O₂ (1:1; flow rate 2 liters/min) for the duration of the experiment.N₂O/O₂ exposure produces a transient state of tetrahydrofolatedeficiency in the rat resulting in formate accumulation followingmethanol administration (J. T. Eells, et al., supra, 2000). In thepresent studies, methanol (25% w/v methanol in saline) was administered(i.p.) to N₂O/O₂ treated rats at an initial dose of dose 4 g/kg,followed by supplemental doses of 1.5 g/kg at 24 and 48 hours. Thismethanol intoxication protocol has been shown to produce a state ofprolonged formic acidemia with formate concentrations between 5-8 mM inmethanol-intoxicated rats resulting in visual dysfunction (M. T. Seme,et al., supra, 1999; M. T. Seme, et al., supra, 2001). Moreover, similarconcentrations of blood formate over similar time periods have beenshown to produce ocular toxicity experimentally in monkeys and have beenassociated with visual toxicity in human methanol intoxication (J. T.Eells, supra, 1992; R. Kavet and K. Nauss, supra, 1990; S. O.lngemansson, supra, 1983). Formate concentrations were determined fromtail vein blood samples by fluorometric analysis as previously described(M. T. Seme, et al., supra, 1999; M. T. Seme, et al., supra, 2001; T.G.Murray, et al., 1991).

Light-Emitting Diode Treatment. GaAIAs light emitting diode (LED) arraysof 670 nm wavelength (LED bandwidth 25-30 nm at 50% power) were obtainedfrom Quantum Devices, Inc. (Barneveld, Wis.). Rats were placed in aplexiglass restraint device (12.7×9×7.6 cm). The LED array waspositioned directly over the animal at a distance of 1 inch, exposingthe entire body. Treatment consisted of irradiation at 670 nm for 2minutes and 24 seconds resulting in a power intensity of 28 mW/cm² andan energy density of 4 joules/cm² at 5, 25 and 50 hours after theinitial dose of methanol. These stimulation parameters (670 nm at anenergy density of 4 J/cm²) had been demonstrated to be beneficial forwound healing, and to stimulate cellular proliferation and cytochromeoxidase activity in cultured visual neurons (H. T. Whelan, et al.,supra, 2001; M. T. T. Wong-Riley, et al., supra, 2001).

ERG Procedures and Analyses. ERG experiments were performed aspreviously described (M. T. Seme, et al., supra, 1999; M. T. Seme, etal., supra, 2001). The light stimulation apparatus consisted of athree-beam optical system. All three beams were derived fromtungsten-halide lamps (50 W, 12 V). Beam intensity was controlled byusing neutral density step filters. ERG recordings were differentiallyamplified and computer-averaged. The amplified signal was processedthrough a two-stage active narrow bandpass filter (the half voltage ofthis filter was 0.2 times the center frequency). To ensure that anytransients in the response that occur at the onset of the stimuluspulses were not included in the average, the initiation of signalaveraging was delayed by a preset number of stimulus cycles (typically aminimum of 20). The resulting ERG is an extremely noise-free, singlecycle, sinusoidal waveform. The averaged responses were measured(peak-to-trough amplitude) from a calibrated digital oscilloscopedisplay.

Before ERG analysis, ophthalmoscopic examination confirmed that all eyeswere free of lenticular opacities or other gross anomalies. Rats wereanesthetized with thiobutabarbitol sodium salt (100 mg/kg, i.p.),positioned in a Kopf stereotaxic apparatus and placed on a heating padto maintain core body temperature at 37° C. Atropine sulfate (0.05mg/kg, s.q.) was administered to inhibit respiratory-tract secretions.The pupil of the eye to be tested was dilated by topical application of1% atropine sulfate. Methylcellulose was topically applied as alubricant and to enhance electrical conduction. A circular silver, wirerecording electrode was positioned on the cornea, a reference electrodewas placed above the eye, and a ground electrode was placed on thetongue. Recordings were obtained under ambient light conditions fromcool white fluorescent room lights approximately 100 cd/m² at the rat'seye. Flickering stimuli (light/dark ratio=0.25:0.75) were presented.Responses to 60 successive flashes were averaged for each stimuluscondition. At each test wavelength, a minimum of four stimulusintensities spaced at intervals of 0.3 log unit, were presented. Thestimulus intensity yielding a 5-μV criterion response was determined byextrapolating between the two intensity points that bracketed the 5-μVresponse for each animal. All sensitivity measures were made intriplicate.

Two experimental protocols were used to evaluate retinal function. (J.F. Rizzo, supra, 1995)

15 Hz/510 nm ERG Response. ERGs were recorded in response to a 15-Hzflickering light at a wavelength of 510 nm over a 3-log unit range oflight intensity. For these studies, the unattenuated stimulus (logrelative retinal illumination=0) had an irradiance of 25 μW distributedover the 70° patch of illuminated retina. This can be calculated toproduce retinal illumination equivalent to about 10⁴ scotopic trolands.These recording conditions disadvantage rods; however, since at least97% of rat photoreceptors are rods and ERGs are recorded at luminanceintensities ranging from 10¹ to 10⁴ scotopic trolands, it is likely thatthe responses to the 15 Hz/510 nm light are drawn from both rods andmedium wavelength cones (M-cones) (M. T. Seme, et al., supra, 1999; M.T. Seme, et al., supra, 2001; D. A. Fox and L. Katz, Vision Res.32:249-255, 1992).

25 Hz/UV ERG Response. UV-sensitive cone responses were elicited by a25-Hz flickering ultraviolet light (380-nm cut off) in the presence ofan intense chromatic adapting light (445 μW) which eliminated responsesmediated by rods and M-cones (G. Jacobs, et al., Nature 353:655-656,1991). The 25-Hz/UV ERG responses were recorded over a 1.5-log unitrange of light intensity. For these studies, the unattenuated stimulus(log relative retinal illumination=0) had an irradiance of 25 μWdistributed over the 70° patch of illuminated retina. This can becalculated to produce retinal illumination equivalent to about 10⁴scotopic trolands in the rat eye.

Histopathologic Analysis. Retinal tissue was prepared for histology aspreviously described (M. T. Seme, et al., supra, 1999; M. T. Seme, etal., supra, 2001). Thick sections (1 μ) for light microscopy werestained with toluidine blue; thin sections for electron microscopy werestained for uranyl acetate-lead citrate (M. T. Seme, et al., supra,1999; M. T. Seme, et al., supra, 2001).

Statistical Analysis. All values are expressed as means±SEM. A one-wayANOVA with Bonferroni's test was used to determine whether anysignificant differences existed among groups for blood formateconcentrations. For ERG studies, a two-way ANOVA was performed. In allcases, the minimum level of significance was taken as P<0.05.

Results

Blood formate accumulation in methanol-intoxicated rats is not alteredby 670 nm LED treatment. Formic acid is the toxic metabolite responsiblefor the retinal and optic nerve toxicity produced in methanolintoxication (G. Martin-Amat, et al., supra, 1977; J. T. Eells, supra,1992; J. T. Eells, et al., supra, 2000; G. Martin-Amat, al., supra,1978). Linear increases in blood formate concentrations were observed inboth methanol-intoxicated and LED-treated methanol-intoxicated ratsduring the 72-hour intoxication period (FIG. 1).

Referring to FIG. 1, photobiomodulation does not alter blood formateconcentrations in methanol-intoxicated rats. Blood formateconcentrations were determined before methanol administration and at 24hour intervals after methanol administration for 72 hours. Shown are themean values±SEM from six rats in each experimental group. Blood formateconcentrations did not differ between the methanol-intoxicated andLED-treated, methanol-intoxicated groups (P>0.05).

In both treatment groups, blood formate concentrations increased tenfoldfrom endogenous concentrations of 0.5-0.6 mM prior to methanoladministration to nearly 6 mM following 72 hours of intoxication. Therate of formate accumulation and blood formate concentrations did notdiffer between the two treatment groups, indicating that LED treatmentdid not alter methanol or formate toxicokinetics. Similar increases inblood formate have been shown to disrupt retinal function in methanolintoxicated rats (M. T. Seme, et al., supra, 1999; M. T. Seme, et al.,supra, 2001) and have been associated with visual toxicity in humanmethanol intoxication (J. T. Eells, supra, 1992; R. Kavet and K. Nauss,supra, 1990).

Methanol-induced retinal dysfunction is attenuated by 670-nm LEDtreatment. Following 72 hours of methanol intoxication, the function ofrods and M-cones was assessed by recording the retinal response to a15-Hz flickering light at wavelength of 510 nm (M. T. Seme, et al.,supra, 1999; M. T. Seme, et al., supra, 2001).

Referring to FIG. 2, photobiomodulation improves rod and M-cone ERGresponse in methanol-intoxicated rats. Rod and M-cone (15 Hz/510 nm) ERGanalysis was performed after 72 hours of methanol intoxication. Shownare the mean values±SEM from six rats in the untreated control,methanol-intoxicated, and LED-treated, methanol-intoxicated experimentalgroups and four rats from the LED control group. ERG responses inmethanol-intoxicated and LED-treated, methanol-intoxicated rats weresignificantly lower than those measured in control rats (*, P<0.001).ERG responses in LED-treated, methanol-intoxicated rats weresignificantly greater than those measured in methanol-intoxicated rats(∓, P<0.001).

In the untreated control group, 15-Hz/510-nm ERG amplitude increasedlinearly over the 3-log unit range of retinal illumination intensities,achieving a maximal amplitude of 65±5 μV at maximal retinal illumination(0 log relative retinal illumination (LRRI) equivalent to 10⁴ scotopictrolands). A similar ERG response profile was observed in LED-controlanimals. In both control groups a consistent 5-μV criterion thresholdresponse was obtained at −3.0±0.1 LRRI. In agreement with our previousstudies, methanol intoxication produced a profound decrease in retinalsensitivity to light coupled with an attenuation of maximal ERG responseamplitude (M. T. Seme, et al., supra, 1999; M. T. Seme, et al., supra,2001). The light intensity required to elicit a threshold (5 μV)15-Hz/510-nm ERG response was increased by 0.6 log units to −2.4±0.1LRRI in methanol-intoxicated rats relative to control animals. Inaddition, the amplitudes of the flicker ERG responses were significantlyattenuated at all luminance intensities achieving a maximal amplitude of18±5 μV, approximately 28% of the maximum control response. Thesechanges are indicative of a severe deficit in retinal function and areconsistent with formate-induced inhibition of photoreceptor oxidativemetabolism (M. T. Seme, et al., supra, 1999; M. T. Seme, et al., supra,2001; G. Jacobs, et al., supra, 1991; A. Koskelainen, et al, Vision Res.34:983-994, 1994; O. Findl, et al., Invest. Ophthalmol. Visual Sci.36:1019-1026, 1995). LED treatment significantly improved rod and M-conemediated ERG responses in methanol intoxicated rats. At lower luminanceintensities (<1.5 LRRI), LED treatment had no effect on ERG response;however, at luminance intensities >1.5 LRRI, ERG responses weresignificantly greater in LED-treated rats compared to methanolintoxicated animals. The maximal rod and M-cone in LED-treated rats was47±8 μV, 72% of the maximal control response. These data are indicativeof a partial recovery of rod and M-cone function by LEDphotobiomodulation in methanol-intoxicated rats.

The function of UV-sensitive cones was examined by recording the retinalresponse to a 25-Hz flickering ultraviolet light (380-nm cutoff) in thepresence of an intense chromatic adapting light. These conditions havebeen shown to isolate the UV-cone response in the rat retina (G. Jacobs,et al., supra, 1991). The effects of methanol intoxication and LED lighttreatment on UV-cone ERG responses are shown in FIG. 3.

Referring to FIG. 3, photobiomodulation improves UV-cone ERG response inmethanol-intoxicated rats. UV-cone (25 Hz/380 nm) ERG analysis wasperformed after 72 hours of methanol intoxication. Shown are the meanvalues±SEM from six rats in the control, methanol-intoxicated, andLED-treated, methanol-intoxicated experimental groups and four rats fromthe LED control group. UV-cone ERG responses were recorded from the sameanimals in which the rod and M-cone responses were recorded. ERGresponses in methanol-intoxicated and LED-treated, methanol-intoxicatedrats were significantly lower than those measured in control rats (*,P<0.001). ERG responses in LED-treated, methanol-intoxicated rats weresignificantly greater than those measured in methanol-intoxicated rats(∓, P<0.05).

In untreated control animals the UV-cone-mediated ERG amplitudeincreased linearly from a 5-μV threshold value (−1.4±0.03 LRRI) to amaximal value of 56±3 μV over the 1.5-log unit range of retinalillumination used in these studies. LED-treated control animalsexhibited a similar ERG response profile to that observed in untreatedcontrol animals. In methanol-intoxicated rats, the UV-cone ERG responsewas profoundly attenuated consistent with our previous studies (M. T.Seme, et al., supra, 1999; M. T. Seme, et al., supra, 2001). The lightintensity required to elicit a 5-μV response was increased by 0.5 logunits to 0.9±0.08 LRRI in intoxicated animals, and the maximal responseamplitude was reduced to 18±6 μV, 30% of the maximum control response.Similar to what we observed in the rod and M-cone ERG studies, LEDtreatment had no effect on UV-cone ERG response at lower luminanceintensities, but significantly improved ERG response at higher luminanceintensities. The maximal UV-cone ERG response in LED-treated rats was37±7 μV, 61% of the control response indicative of a partial recovery ofUV-cone function by LED photobiomodulation.

Methanol-induced retinal histopathology is prevented by 670 nm LEDtreatment. The architecture of the retina in methanol-intoxicated andLED-treated methanol intoxicated rats was evaluated by light andelectron microscopy. These studies focused on the outer retina at thelevel of the photoreceptors based on our previous findings of outerretinal pathology and photoreceptor mitochondrial disruption followingmethanol intoxication (M. T. Seme, et al., supra, 1999; M. T. Seme, etal., supra, 2001; T. G. Murray, et al., supra, 1991). FIG. 4 illustratesouter retinal morphology in representative untreated control (FIG. 4A),LED control (FIG. 4B), methanol intoxicated (FIG. 4C), and LED-treatedmethanol intoxicated (FIG. 4D) retinas.

Referring to FIG. 4, photobiomodulation protects retinal morphology inmethanol-intoxicated rats. Outer retinal morphology in representativeuntreated control (A), LED control (B), methanol-intoxicated (C), andLED-treated, methanol-intoxicated (D) rats. Sections were taken from theposterior pole of the retina within two disk diameters of the opticnerve in any direction. (Toluidine blue, ×450.) (A) rpe, retinal pigmentepithelium; os, photoreceptor outer segments; is, photoreceptor innersegments; onl, outer nuclear layer; opl, outer plexiform layer; ipl,inner plexiform layer. (B) The arrows indicate enlargement and swellingof the photoreceptor inner segments, and the circles indicate thefragmented appearance of photoreceptor nuclei. No histopathologicchanges were apparent at the light microscopic level in the LED controlor LED-treated, methanol-intoxicated groups.

Pronounced histopathologic changes were apparent in the outer retina ofmethanol-intoxicated rats (FIG. 4C), including evidence of retinaledema, swelling of photoreceptor inner segments, and morphologic changesin photoreceptor nuclei. Retinal edema was evidenced by the spacingbetween the photoreceptor inner segments, and by the spacing of thenuclei in the outer nuclear layer. Photoreceptor inner segments wereprofoundly swollen and enlarged, and photoreceptor nuclei in the outernuclear layer appeared fragmented with irregularly stained chromatin. Incontrast, LED-treated methanol-intoxicated animals (FIG. 4D) exhibitedretinal morphology which was indistinguishable from untreated controlrats (FIG. 4A) and LED treated control rats (FIG. 4B). In these animalsouter retinal morphology was characterized by ordered photoreceptorinner segments with no evidence of vacuolization or swelling and theouter nuclear layer was compact with round and well-defined nuclei. Thelack of retinal histopathology in LED-treated methanol intoxicated ratsprovides additional evidence of the retinoprotective actions of 670-nmLED treatment.

The most obvious ultrastructural change observed in the outer retina ofmethanol-intoxicated rats was swelling and disruption of mitochondria inthe inner segments of the photoreceptors. Referring to FIG. 5,photobiomodulation protects photoreceptor ultrastructure inmethanol-intoxicated rats. Electron micrographs of the rod inner segmentregion in representative untreated control (A), LED control (B),methanol-intoxicated (C), and LED-treated, methanol-intoxicated (D)rats. The arrows indicate abnormal mitochondrial morphology inphotoreceptor inner segments. Photoreceptor mitochondria from LEDcontrol or LED-treated, methanol- intoxicated rats exhibited normalmorphology with well-defined cristae, (Magnifications: ×5,000).

Some mitochondria were swollen and contained expanded cristae; othermitochondria were disrupted and showed no evidence of cristae (FIG. 5C).In contrast, mitochondria in the photoreceptor inner segments fromLED-treated, methanol-intoxicated rats (FIG. 5D) exhibited normalmorphology with well-defined cristae similar to inner segmentmitochondrial morphology in untreated control rats (FIG. 5A) andLED-treated control rats (FIG. 5B). The absence of mitochondrial damagein photoreceptors of LED-treated methanol-intoxicated rats stronglysupports our hypothesis that 670-nm LED treatment preservedmitochondrial function.

Discussion

Low-energy photon irradiation by light in the far-red to near-IRspectral range 630-1000 nm) using low-energy lasers or LED arrays hasbeen found to modulate various biological processes in cell culture andanimal models (M. J. Conlan, et al., supra, 1996; W. Yu, et al., supra,1997; A. P. Sommer, et al., supra, 2001; T. Karu, supra, 1999). Thisphenomenon of photobiomodulation has been applied clinically in thetreatment soft tissue injuries and to accelerate wound healing (H. T.Whelan, et al., supra, 2001; M. J. Conlan, et al., supra, 1996). Themechanism of photobiomodulation by red to near-IR light at the cellularlevel has been ascribed to the activation of mitochondrial respiratorychain components, resulting in initiation of a signaling cascade whichpromotes cellular proliferation and cytoprotecton (T. Karu, supra, 1999;N. Grossman, et al., supra, 1998; M. T. T. Wong-Riley, et al., supra,2001). A comparison of the action spectrum for cellular proliferationafter photoirradiation with the absorption spectrum of potentialphotoacceptors lead Karu (T. Karu, supra, 1999) to suggest thatcytochrome oxidase is a primary photoreceptor of light in the red tonear-IR region of the spectrum.

Recent studies conducted in primary neuronal cultures by our researchgroup have shown that 670-nm LED photobiomodulation reversed thereduction in cytochrome oxidase activity produced by the blockade ofvoltage-dependent sodium channel function by tetrodotoxin andup-regulated cytochrome oxidase activity in normal neurons (M. T. T.Wong-Riley, et al., supra, 2001). The present studies extended theseinvestigations to an in vivo system to determine if 670-nm LEDphotobiomodulation would improve retinal function in an animal model offormate-induced mitochondrial dysfunction. Results of this studydemonstrate the therapeutic benefit of photobiomodulation in thesurvival and functional recovery of the retina in vivo after acuteinjury by the mitochondrial toxin, formic acid generated in the courseof methanol intoxication. We provide in vivo evidence that three briefpost-methanol-intoxication treatments with 670-nm LED photoirradiationpromotes the recovery of retinal function in rod and cone pathways andprotects the retina from the histopathologic changes induced bymethanol-derived formate. These findings provide a link between theactions of red to near-IR light on mitochondrial oxidative metabolism invitro and retinoprotection in vivo.

Low-energy laser irradiation has documented benefits in promoting thehealing of hypoxic, ischemic, and infected wounds (H. T. Whelan, et al.,supra, 2001; M. J. Conlan, et al., supra, 1996). However, lasers havelimitations in beam width, wavelength capabilities, and size of woundsthat can be treated (H. T. Whelan, et al., supra, 2001). Heat generatedfrom the laser light can damage biological tissue, and the concentratedbeam of laser light may accidentally damage the eye. LED arrays weredeveloped for National Aeronautics and Space Administration manned spaceflight experiments. In comparison to lasers, the patented LED technologygenerates negligible amounts of heat, is clinically proven to be safe,and has achieved non-significant risk status for human trials by theFood and Drug Administration (H. T. Whelan, et al., supra, 2001). Thewavelength, power, and energy parameters used in the present study arebased on their beneficial effects for wound healing in humans (H. T.Whelan, et al., supra, 2001) and stimulation of CO activity in culturedneuronal cells (M. T. T. Wong-Riley, et al., supra, 2001).

The retinoprotective actions of 670-nm LED treatment in the presentstudy are consistent with the actions of formate as a mitochondrialtoxin and the actions of 670-nm light on cytochrome oxidase activity.Formate has been shown to reversibly inhibit cytochrome oxidase activitywith an apparent inhibition constant between 5 and 30 mM (P. Nicholls,supra, 1975; P. Nicholls, supra, 1976). Blood formate concentrations inmethanol-intoxicated rats in the present study fall within this rangeand retinal formate concentrations closely parallel blood formateconcentrations (J. T. Eells, et al., supra, 2000). The functional andmorphologic alterations produced in the retina by methanol-derivedformate are indicative of formate-induced inhibition of photoreceptormitochondrial energy metabolism. Photoreceptors are the mostmetabolically active cells in the body, and the energy required forphototransduction is derived primarily from oxidative metabolism (A.Ames, III, et al., J. Neurosci. 12:840-853, 1992; A. Ames, III, Can. J.Physiol. Pharmacol. 70:S158-S164, 1992). The loss of retinal sensitivityto light and attenuation of ERG response in methanol-intoxicated ratsare indicative of formate-induced inhibition of photoreceptor oxidativeenergy metabolism and are similar to the actions of other metabolicpoisons in the retina (M. T. Seme, et al., supra, 1999; A. Koskelainen,et al., supra, 1994; O. Findl, et al., supra, 1995). The observedmitochondrial swelling and disruption in the photoreceptor innersegments in methanol-intoxicated rats are consistent with a disruptionof ionic homeostasis secondary to inhibition of cytochrome oxidase.Moreover, similar morphologic alterations have been reported in theretinas of patients with mitochondrial diseases that inhibit electrontransport (P. A. McKelvie, et al., J. Neurol. Sci. 102:51-60, 1991; L.M. Rapp, etal., Invest. Ophthalmol. Visual Sci. 31:1186-1190, 1990; P.Runge, et al., Br. J. Opththalmol. 70:782-796, 1986).

In the present study, the increase in ERG response and the lack ofdamage to photoreceptor mitochondria in LED-treated,methanol-intoxicated rats are indicative of a biostimulatory effect of670-nm light on photoreceptor bioenergetics. A growing body of evidencesuggests that cytochrome oxidase is a key photoacceptor of light in thefar red to near infrared spectral range (T. Karu, supra, 1999; W. Yu, etal., supra, 1997; S. Passarella, et al., FEBS Lett. 175:95-99, 1984; D.Pastore, et al., Int. J. Radiat. Biol. 76:863-870, 2000). Cytochromeoxidase is an integral membrane protein which contains four redox activemetal centers and has a strong absorbance in the far-red to near-IRspectral range detectable in vivo by near-IR spectroscopy (C. E. Cooperand R. Springett, Philos. Trans. R. Soc. London B352:9-676, 1977; B.Beauvoit, et al., Anal. Biochem. 226:167-174, 1995; B. Beauvoit, et al.,Biophys. J. 67:2501-2510, 1994). Moreover, 660-680 nm irradiation hasbeen shown to increase electron transfer in purified cytochrome oxidase(D. Pastore, et al., supra, 2000), increase mitochondrial respirationand ATP synthesis in isolated mitochondria (S. Passarella, et al.,supra, 1984), and to up regulate cytochrome oxidase activity in culturedneuronal cells (M. T. T. Wong-Riley, et al., supra, 2001). Anup-regulation of retinal cytochrome oxidase by LED treatment wouldeffectively counteract the inhibitory actions of formate on retinaloxidative metabolism, thus improving retinal function. Although retinalfunction was improved in LED-treated rats, it was not restored tocontrol response levels. At lower luminance intensities, LED treatmentdid not improve the ERG response in methanol-intoxicated rats suggestingthat the rate of activation of some components of phototransductionactivation remained compromised by formate. Because the rate ofactivation of phototransduction depends on an adequate supply of GTP andATP (G. Jacobs, et al., supra, 1991; A. Koskelainen, et al., supra,1994; O. Findl, et al., supra, 1995), it is possible that theformate-induced metabolic inhibition is only partly attenuated by ourLED treatment protocol.

The prolonged effect of three brief LED treatments in mediating theretinoprotective actions in methanol intoxication suggests that 670-nmLED photostimulation induces a cascade of signaling events initiated bythe initial absorption of light by cytochrome oxidase. These signalingevents may include the activation of immediate early genes,transcription factors, cytochrome oxidase subunit gene expression, and ahost of other enzymes and pathways related to increased oxidativemetabolism (T. Karu, supra, 1999; M. T. T. Wong-Riley, et al., supra,2001; C. Zhang and M. Wong-Riley, Eur. J. Neurosci. 12:1013-1023, 2000).In addition to increased oxidative metabolism, red to near-IR lightstimulation of mitochondrial electron transfer is also known to increasethe generation of reactive oxygen species (T. Karu, supra, 1999). Thesemitochondrially generated reactive oxygen species may function assignaling molecules to provide communication between mitochondria andthe cytosol and nucleus and thus play an important signaling role in theactivation of retinoprotective processes following LED treatment (S.Nemoto, et al., Mol. Cell. Biol. 20:7311-7318, 2000).

The results of this study demonstrate that photobiomodulation with redto near-IR light augments recovery pathways promoting neuronal viabilityand restoring neuronal function following injury. Importantly, there wasno evidence of damage to the normal retina following 670-nm LEDtreatment. Based on these findings, we propose that photobiomodulationrepresents an innovative and novel therapeutic approach for thetreatment of retinal injury, as well as the treatment of retinaldiseases including age-related macular degeneration, glaucoma, diabeticretinopathy and Leber's hereditary optic neuropathy.

EXAMPLE 2 Animal Model of Retinal Protection.

Methanol intoxication produces toxic injury to the retina and opticnerve frequently resulting in blindness. The toxic metabolite inmethanol intoxication is formic acid, a mitochondrial toxin known toinhibit the essential mitochondrial enzyme, cytochrome oxidase. TheEells' laboratory has developed a rodent model of methanol toxicitywhich replicates the metabolic and retinotoxic manifestations of humanmethanol toxicity. This animal model also manifests many featuresassociated with retinal aging and many clinically important retinal andoptic nerve diseases and thus serves as an excellent experimental modelfor the investigation of treatments for retinal and optic nerve disease.

Studies were undertaken to determine if exposure to monochromatic 670 nmradiation from light-emitting diode (LED) arrays would protect theretina against the toxic actions of methanol-derived formic acid in thisanimal model of ocular disease. Methanol-intoxicated and non-intoxicatedcontrol rats were placed in a plexiglass restraint device (12.7×9×7.6cm). The LED array was positioned directly over the animal at a distanceof 2.5 cm. Treatment consisted of irradiation at 670 nm for 2 min and 24sec resulting in a power intensity of 28 mW/cm² and an energy density of4 joules/cm². NIR-LED treatments were administered 5, 25 and 50 hoursafter the initial dose of methanol. These stimulation parameters (670 nmat an energy density of 4 J/cm²) have been demonstrated to be beneficialfor wound healing, and to stimulate cellular proliferation andcytochrome oxidase activity in cultured visual neurons.

The electroretinogram (ERG) which measures the response of the retina toflickering light stimulation was used as a sensitive and clinicallyrelevant indicator of retinal function. NIR-LED treated animalsexhibited a dramatic improvement in retinal function measured by the ERG(FIG. 6) and NIR-LED treatment also protected the retina from thehistopathologic damage induced by methanol-derived formate. Thesefindings provide a link between the actions of monochromatic red to nearinfrared light on mitochondrial oxidative metabolism in vitro andretinoprotection in vivo.

EXAMPLE 3 Nonhuman Primate Model of Retinal Protection.

We have initiated studies of laser retinal injury in a nonhuman primatemodel. To date, we have performed two experiments using this animalmodel. In each experiment one monkey was lased without LED treatment andone lased with LED treatment (670 nm, 4 J/cm²). A laser grid (128 spotsdelivered to the macula and perimacula) was created in the centralretina of right eye of each animal. This grid consisted of grade I andII burns, photocoagulating the photoreceptors and outer nuclear layer ofthe retina. Multifocal ERG was performed to assess the functional stateof the retina. In the first experiment, the LED-treated monkey wastreated at 1, 24, 72 and 96 hours post injury. ERG amplitude in both LEDtreated and untreated monkeys was temporarily increased shortly afterlaser injury and this increase was greater in the LED-treated monkey.Assessment of the severity of the laser burn in LED treated anduntreated animal demonstrated a greater that 50% improvement in thedegree of retinal healing at 1 month post-laser in the LED-treatedmonkey (FIG. 7). In addition, the thickness of the retina measured atthe fovea by optical coherence tomography did not differ from thepre-laser thickness in the LED-treated animal whereas it was 50% thinnerin the untreated animal. Importantly, LED treatment prevented the lossof cytochrome oxidase staining (FIG. 8) in the lateral geniculatenucleus clearly showing that the brain was responding to visual inputfrom the “healed” retina in the LED-treated animal much more effectivelythan in the untreated animal.

In the second study, the LED-treated animal was treated once per day for11 days and mfERG recordings were recorded (FIG. 9). Again, shortlyafter laser injury, the ERG amplitude was temporarily increased in bothLED treated and untreated animals. However, in this experiment theincreases were comparable. At 4 days post laser injury, the mfERGresponses in LED treated and untreated animals had decreased topre-laser amplitudes. However, by day 11 post laser, the mfERG responsein the LED treated monkey was more than 50% greater than that measuredin the untreated (sham) monkey. In both experiments, these preliminaryfindings are indicative of improved retinal healing and visual corticalfunction following LED treatment in laser injured primate model.

EXAMPLE 4 Effect of NIR-LED Treatment in Leber's Hereditary OpticNeuropathy.

The effect of NIR-LED treatment was investigated in the treatment ofLeber's Hereditary Optic Neuropathy (LHON). LHON is a disease caused bya mitochondrial mutation (the most common mutation is in position 11778of the mitochondrial genome) which results in defective mitochondrialenergy production and causes blindness in early adulthood.

NIR-LED treatment was investigated in 6 affected (blind) 11778 LHONmutation carriers in Colatina, Brazil according to the protocol approvedby the Institutional Review Board of San Paolo Federal University. Eachsubject exhibited a profound deficit in central vision. Baseline valuesfor NSE, Humphrey 60° visual fields and nerve fiber analysis wereobtained prior to LED treatment. LED treatment consisted of irradiationat 670 nm for 80 seconds delivered to each eye producing an estimatedenergy density of 4 joules/cm² at the optic nerve head. LED treatmentwas administered once per day for 3 days using handheld LED arrays(Quantum Devices, Barneveld, Wis.) positioned 2.5 cm from each closedeye. Treatment response was assessed 1 day following the third LEDtreatment (day 4) and again on day 10. Two of the NIR-LED-treatedsubjects reported a transient improvement in color vision and visualacuity lasting approximately one day. NSE concentrations in these twosubjects increased dramatically (from a pre-exposure level of 0.9 μg/Lto 7.6 μg/L in one subject and 2.2 μg/L to 5.3 μg/L in the other) incontrast to smaller increases or decreases in NSE measured in the otherfour subjects. Peripheral visual fields showed distinct improvement in 4of the 6 patients by 10 days post treatment. No change was observed innerve fiber layer measurements. No detrimental effects of NIR-LEDtreatment were reported by study subjects and no adverse effects wereobserved in visual function tests. The findings of this pilot studyconfirm and extend previous studies which have reported that NIR-LEDexposure at energy densities up to 300 joules/cm² produces nodetrimental effects on the retina and optic nerve. The studies furtherdemonstrate that NIR-LED treatment exerts a beneficial effect in LHON.

1. A method of treating visual system disease or injury, comprising thesteps of a) exposing a component of a patient's visual system to lighttreatment, wherein the light treatment is characterized by wavelengthbetween 630-1000 nm and power intensity between 10-90 mW/cm² for a timeof 1-3 minutes, wherein the treatments are administered at least 2-3times per day and b) observing restoration or protection of visualfunction, wherein the number of metabolically active neurons hasincreased.
 2. The method of claim 1 wherein the wavelength is selectedfrom the group consisting of 670 nm, 830 nm and 880 nm.
 3. The method ofclaim 1 wherein the wavelength is between 670-900 nm.
 4. The method ofclaim 1 wherein the light treatment is characterized by an energydensity of between 0.5-20 J/cm².
 5. The method of claim 4 when theenergy density is between 2-10 J/cm².
 6. The method of claim 1 whereinthe patient is exposed to light treatment multiple times.
 7. The methodof claim 6 wherein the exposure is at least 3 times.
 8. The method ofclaim 1 wherein the patient is exposed to light treatment intervals of24 hours.
 9. The method of claim 1 wherein the component of the visualsystem comprises the patient's retina.
 10. The method of claim 1 whereinthe component of the visual system is selected from the group consistingof cornea and optic nerve.
 11. The method of claim 1 herein the retinalfunction is evaluated.
 12. The method of claim 1 wherein the light issupplied by an LED device.
 13. The method of claim 1 wherein the powerintensity is between 25-50 mW/cm².
 14. A method of treating visualsystem disease or injury, comprising the steps of a) exposing acomponent of a patient's visual system to light treatment, wherein thelight treatment is characterized by wavelength between 630-1000 nm andpower intensity between 10-90 mW/cm² for a time of 1-3 minutes, whereinthe treatments are administered at least 2-3 times per day, and b)observing restoration or protection of visual function, wherein the ofan exogenous photosensitizer exposure is in the absence of aphotosensitizer.
 15. A method of treating visual system disease orinjury, comprising the steps of a) exposing a component of a patient'svisual system to light treatment, wherein the light treatment ischaracterized by wavelength between 630-1000 nm and power intensitybetween 10-90 mW/cm² for a time of 1-3 minutes, wherein the treatmentsare administered at least 2-3 times per day, and b) observingrestoration or protection of visual function, wherein rod and cone ERGamplitude has increased relative to tissue that hasn't been exposed tothe light treatment.